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Year : 2019  |  Volume : 7  |  Issue : 2  |  Page : 56-60

Microbial investigations of fungal infections: An overview

1 Department of Oral Pathology, SJM Dental College and Hospital, Chitradurga, Karnataka, India
2 Department of Oral Pathology, Sharavathi Dental College and Hospital, Shimoga, Karnataka, India
3 Department of Dentistry, Kanachur Institute of Medical Sciences, Natekal, Karnataka, India
4 Department of Oral and Maxillofacial Pathology, Srinivas Institute of Dental Sciences, Surathkal, Mangalore, Karnataka, India

Date of Web Publication29-Aug-2019

Correspondence Address:
Dr. Frankantony P Britto
Department of Oral Pathology, SJM Dental College and Hospital, Chitradurga 577501, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/INJO.INJO_18_19

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The incidence of fungal infections has increased considerably in recent years. Fungal infections remain an important cause of morbidity and mortality. New diagnostic approaches have been developed based on non-culture-based methods, which may allow early diagnosis and treatment of fungal infections. Laboratory procedures in diagnostic mycology are directed mainly toward the direct demonstration of the pathogenic fungi in clinical specimens by microscopy along with successful isolation of pathogenic fungi by using various culture techniques. They also help in the prediction of possible therapeutic outcome by determining antifungal susceptibility and can also be used in epidemiological studies by tracing the source of infection. An active interaction between the clinician, microbiologist, and pathologist facilitates the exact diagnosis and accurate interpretation of culture and histopathological results. This article focuses on specific conventional and rapid advanced techniques concerning a practical approach to the lab investigations in fungal infections.

Keywords: Candida species, culture media, fungal infections, lab investigations

How to cite this article:
Britto FP, Dmello.A XP, Shetty AS, Thakkilipati HC, Udupa R, Shetty J VR. Microbial investigations of fungal infections: An overview. Int J Oral Care Res 2019;7:56-60

How to cite this URL:
Britto FP, Dmello.A XP, Shetty AS, Thakkilipati HC, Udupa R, Shetty J VR. Microbial investigations of fungal infections: An overview. Int J Oral Care Res [serial online] 2019 [cited 2021 Dec 5];7:56-60. Available from: https://www.ijocr.org/text.asp?2019/7/2/56/262310

  Introduction Top

The fungi are saprophytic (derives nourishment from dead organic matter) and parasitic eukaryotic organisms. Although formerly considered to be plants, they are now generally assigned their own kingdom, Mycota.[1] They resemble plants because they have rigid cell walls and are nonmotile. Unlike plants, the fungi lack chlorophyll and are unable to photosynthesize. The fungi also lack the multicellular complexity and organization of most animals.[2]

Opportunistic invasive fungal infections remain an important cause of morbidity and mortality. The most common fungi that cause disease in transplant recipients and other immunocompromised patients are Candida and Aspergillus species.[3],[4] There are several approaches to manage these infections, including prevention, antifungal prophylaxis, and empiric therapy. The first essential component of the treatment is diagnosis. The diagnosis of deep-seated candidiasis is difficult and often depends primarily on the clinical picture, with definitive diagnosis established by histopathology of visceral tissues.[5],[6]

  Collection and Transport of the Clinical Specimen Top

The diagnosis of fungal infections is dependent entirely on the selection and collection of an appropriate clinical specimen for culture. Many fungal infections are similar clinically to mycobacterial infections, and often the same clinical specimen is cultured for both fungi and mycobacteria.[7],[8] Specimens include skin scrapings, hair, nails, respiratory tract secretions, cerebrospinal fluid (CSF), blood, oral and vaginal specimen, urine, pus, ocular specimen, and tissue bone marrow.[9],[10]

Direct microscopic examination of fungal cells within the clinical specimen is a valuable diagnostic procedure for the following reasons: (1) In many instances, a tentative or even a definitive diagnosis can be made before the growth of fungal cells would be apparent in culture, (2) Observing fungal cells in a clinical specimen may be more valuable as a criterion for diagnosis than isolating in a culture. Preparations for direct examination of clinical specimen include potassium hydroxide (KOH), India ink, and Calcofluor White; in addition, a few staining techniques such as Giemsa and periodic acid–Schiff (PAS) are effective.[11]

Wet Smears: Unstained Preparations with KOH

Patches from the mucous membrane of the mouth, vagina, skin, or nails scrapping, sputum, and so on are collected in a sterile container. These are examined in a KOH wet mount or gram stain. Yeast cells of 4–8 μm with budding mixed pseudohyphae, showing colonization and tissue invasion are significant. For the detection of candida, wet smear microscopy has been positive in the majority of cases but not in all cases with positive culture.[3]

Stained preparations: preparations with KOH

Preparation with KOH clears the tissue and cellular debris from all types of clinical specimens without damaging the fungal cells. This clearing process requires only 5–10min, after which one can observe the fungal morphology as well as the pigment of the fungal cell wall.[12] With a bright-field microscope, one can detect hyphae and yeast cells more readily by closing the aperture of the iris diaphragm to reduce the intensity of light. Brown-walled hyphal cells can be detected without a reduction in light. To highlight the fungal cell walls, Parker Super Chrome Blue-Black Ink can be incorporated into the KOH preparation. Adding the dye however will mask the cell wall pigment of dematiaceous fungi.[13]

Preparations with Calcofluor White and KOH

Calcofluor White is used as a whitening agent in the textile and paper industry. The dye is useful for showing the presence of fungal cells in clinical specimens because it binds to β 1–3, β 1–4 polysaccharides. The dye then fluoresces as it is exposed to the shorter wavelengths of ultraviolet light. A fluorescence microscope is needed for detecting fungal cells prepared with Calcofluor White. Yeast cells, pseudohyphae, and hyphae display a chalk-white or brilliant apple-green fluorescence, depending on filters used, that readily differentiates them from background material. The disadvantages of using Calcofluor are the need for a fluorescence microscope, the inability of the dye to detect the endospores within a spherule of Coccidioides immitis, and the difficulty in interpreting vaginal secretions.[14]

Preparation with India ink

India ink is useful for indicating the presence or absence of extracellular polysaccharide capsules of fungal cells. The technique is particularly helpful for detecting Cryptococcus neoformans in CSF. Because India ink serves as a negative stain, the encapsulated yeast cells can be readily detected against the dark background. The ink should be free from artifacts or granular carbon particles to ensure a good preparation. The presence of encapsulated yeast cells in CSF is almost always an indicator of cryptococcal meningitis. Making the distinction between encapsulated yeasts and artifacts requires considerable experience.[15]

Preparations with PAS stain

The PAS stain is one of the most widely used stains for fungal histopathology. The PAS reaction stains certain polysaccharides found in the fungal cell wall. For good results, both the periodic acid solution and the sodium metabisulfite solution should be fresh and protected from light. For laboratories with a fluorescence microscope, Calcofluor White is preferred over PAS.[6]

Acid-fast stain procedure for Nocardia (modified Kinyoun method)

Acid-fast staining is useful for detecting Nocardia species and for differentiating them from other aerobic actinomycetes. Some of the filaments stain red with carbol-fuchsin staining, whereas others may appear blue because of counterstaining effect.[16]

Gram’s stain

It is effective for some pathogens but not for others. In general, the procedure is more suited to sections than that to smears.

Gomori’s methenamine silver stain (Grocott’s modification)

It is based on the liberation of aldehyde groups and their subsequent identification by the reduced silver method. It is used for the demonstration of polysaccharide content on the fungus in tissue sections. The aldehydes reduce the methenamine silver nitrate complex, resulting in brown-black staining of fungal cell wall because of the deposition of reduced silver wherever aldehydes are located.

Inference: The fungi and bacteria are stained black, mucopolysaccharide dark grey, cytoplasm old rose, and the tissue pale green. The Gomori’s methenamine silver stain is better than other fungal stains as it stains both live and dead fungi in contrast to PAS, which stains only live fungi. It stains the higher bacteria (Nocardia and Actinomyces) also, which are not stained by other fungal stains.[17]

  Culture Medias Top

All fungi require several specific elements for growth and reproduction. The requirements for growth are generally less stringent than that for sporulation, so it is often necessary to try several types of media when attempting to identify a fungus in culture.

Common media for primary fungal isolation include Sabouraud dextrose agar and brain heart infusion agar, either in  Petri dish More Detailses or screwtop tubes. The media may be enriched with 5%–10% sheep blood to support the growth of certain fungi. Specimens that are likely to be contaminated with other microorganisms, such as urine or sputum, are set up on agar media containing antimicrobials. Chloramphenicol, streptomycin, or penicillin are incorporated into the agar to inhibit the growth of bacteria, and cycloheximide is used to inhibit the growth of contaminant. Most fungi also thrive on potato dextrose agar (PDA), but this can be too rich for many fungi, so that excessive mycelial growth is obtained at the expense of sporulation.[18]

Culture media used for fungal growth

The most commonly used culture media for fungal growth are as follows: Sabouraud agar, Hay infusion agar, PDA, potato dextrose broth, yeast agar and broth, mycological agar, malt extract agar and broth, soy peptone yeast extract agar, water agar, antibiotic agar, acidified cornmeal agar, cornmeal agar, and potato carrot agar.[19],[20]

Germ tube formation (Reynolds-Braude phenomenon)

The serum germ tube test is a rapid “presumptive test” for Candida albicans. A light inoculum of cultured yeast is incubated in bovine serum for 2–3h at 37°C. The test is positive if there are short hyphae without a constriction where the hypha joins the parent cell. Strains of C. albicans produce germ tubes from their yeast cells when placed in a liquid nutrient environment and incubated at 35°C for 3h. It is simple, efficient, most accepted and economic test for screening and rapid identification for C. albicans. True germ tube is defined as hyphal projections from the germinating yeast cell, lacking any constriction at the point of origin. It is important to read the test within 3h as other candidal species will also form germ tubes in serum after this period. In conclusion, germ tube appears to be the cheapest test but it is time-consuming and laborious.[21]

Adhesive (Scotch) tape preparation

It is carried out by touching the adhesive side of a small length of transparent tape to the surface of the colony. Adhere the length of the tape to the surface of a microscope slide to which a drop of lactophenol cotton or aniline blue has been added. Observe microscopically for the characteristic shape and arrangement of the spores. The transparent adhesive tape preparation allows one to observe the organism microscopically approximately the way it sporulates in culture. The spores are usually intact, and the microscopic identification of an organism can be made easily. Some laboratories prefer to use the microslide culture for making the microscopic identification of an organism.[22]

  Biochemical Tests Top

The different biochemical tests are as follows:

Carbohydrate fermentation

Sugars are vital to all living organisms. Yeast is capable of using some but not all sugars as a food source. Yeast can metabolize sugar in two ways, aerobically, with the aid of oxygen, or anaerobically, without oxygen.

Carbohydrate assimilation

This test is used for definite speciation of Candida and few other fungal microorganisms. In the carbohydrate assimilation test (modified Wickerham method), the carbohydrates used are glucose, maltose, lactose, sucrose, galactose, xylose, trehalose, and cellobiose.[23]

Rapid urease test

This test is used to detect the presence of urease enzyme produced by different Candida species. Christensen’s urea agar slants are used. Conversion of the yellow slope to pink or red is considered positive. A negative test is reported when no color change is observed. Other techniques are microslide culture, hair preformation test, in vitro conversion of dimorphic molds, exo-antigen test, and nucleic acid probe testing.[12]

Newer diagnostic techniques are computed tomography scanning, magnetic resonance imaging techniques, nuclear magnetic resonance, immunodiagnostic techniques, polymerase chain reaction (PCR) techniques, serologic test, latex agglutination test, and immunofluorescence.[24]

  Immunodiagnosis Top

The basis of immunodiagnosis is the patient’s response to candida, as expressed by the presence of antibodies or cell mediated immunity (CMI) or the presence of fungal antigens in the patient’s body fluids. As a consequence, different types of assays are involved in immunodiagnosis.[25]

Test for detection of antibodies

The tests used to detect antibodies of candida are agglutination of latex-coated particle, immunodiffusion, immune electrophoretic test systems, radioimmunoassay, and enzyme immunoassay. For the detection of antibodies diagnostic for a candidal infection, various candidal structures have been tried as antigens: cell wall mannan, glucan polysaccharides, mixed cytoplasmic antigen preparation, and antigens expressed exclusively by the hyphal forms of C. albicans. The most promoting antigens have been purified enolase as it is a protein expressed at high levels in C. albicans cytosol, HSP90, and stress protein. False-negative results are the major problem of these tests because of less sensitivity and noncompetent immune status of patients with candida.[25]

Tests for detection of antigen to Candida species

The various antigens detected by different systems are as following:

  1. Detection of mannan using enzyme immunoassays such as Pastorex latex agglutination systems, incorporating monoclonal antibodies, and candida antigen detection systems, incorporating polyclonal antibodies.

  2. Detection of glycoprotein antigen using Cand-Tec latex agglutination test.

  3. Detection of 47–49kDa protein, apparently enolase using enzyme-linked immunosorbent assay.

Antigens are detected generally in serum. However, they can also be found in other body fluids, including urine. The antigen detection tests are more significant for diagnosis because they detect active infection. But they are not sensitive enough and hence lead to false-negative results. Another false-negative result may come from cases where the infective agent is one of the rare Candida species, which are not recognized by the detecting reagents.[2]

Detection of cell-mediated immunity

These are valuable in assessing immunocompetence of patients but not for the diagnosis of candidiasis.[2] The tests used for the detection of CMI are in vivo tests such as skin test, in vitro tests such as lymphocyte transformation test.

Other tests

Detection of fungal metabolites: d-arabitol is produced by most species of Candida except Candida krusei and Candida glabrata. In serum and urine, it can be detected by gas liquid chromatography and enzymatic fluorometric method. Serum arabitol levels can be elevated in patients with renal insufficiency, mannitol interference, and steroid therapy. These tests, hence, give false-positive results. To overcome this problem, determination of serum arabitol/creatinine ratio has been advised.[2]

Molecular biology techniques: Use of specific DNA probes, such as P450, 14-lanosterol demethylase, parts of 18S rRNA gene complex, chitin synthetase gene, and mitochondrial DNA, has proved to be useful.[26]

RNA profiling: Can be carried out by doing electrophoresis using different techniques such as contour clamped homogenous electric field, orthogonal field agarose gel electrophoresis or field invasion gel electrophoresis, restriction enzyme analysis, and DNA amplification techniques such as PCR.[27]

Histopathology: Incisional and excisional biopsies are useful in the diagnosis of persistent oral white lesions, which are thought to be related to candida infection. As a significant proportion of chronic candida leukoplakic lesions are premalignant, a biopsy in addition to a swab is essential if the lesion does not resolve after antifungal therapy.[28]

  Conclusion Top

Key to successful laboratory diagnosis includes recognizing the likely causative agents, determining the best specimen type for investigation, avoiding contamination from commensal organisms during specimen collection, and communication with the clinical microbiology laboratory regarding specimen collection, transport, and testing for suspected pathogens.

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Conflicts of interest

There are no conflicts of interest.

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